Salmonellae are Enterobacteriaceae that cause a spectrum of diseases in humans and animals, including enteric (typhoid) fever and gastroenteritis. Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening systemic disease that is responsible for significant morbidity and mortality annually worldwide. Approximately 3-5% of individuals infected with S. Typhi become chronic carriers with the gallbladder (GB) as the site of persistence. S. Typhi is a human-restricted pathogen, therefore asymptomatic carriers represent a critical reservoir for further spread of disease. We have demonstrated that gallstones aid in the development and maintenance of GB carriage in a mouse model and in humans, serving as a substrate to which salmonellae attach and form a protective biofilm. However, the molecular basis of chronic carriage of Salmonella in the GB, both from the host and bacterial perspectives, is poorly understood. Our goal is to better understand the environment that allows for asymptomatic chronic carriage and to develop therapies to reverse/prevent it. In Aim 1 of this application, we will examine the ongoing dynamics of the GB response to Salmonella colonization in our gallstone mouse model to better understand the role of the host in permitting asymptomatic chronic carriage. We will focus on the role of immune factors in this process, including IL-10, which we have demonstrated to participate in the development of GB chronic colonization. In Aim 2, we examine the bacterium throughout the establishment of chronic infection, including genes specifically expressed in carriers and putative GB-driven pathoadaptive mutations. In Aim 3, we expand the development of novel treatments for chronic carriage that have emerged from our discovery research efforts. These approaches target kinases required for biofilm formation and the gallstone itself. This work will identify key host and bacterial factors at play during chronic infection of the GB by Salmonella, thus presenting new targets for therapeutic/preventive approaches to eliminate the carrier state.